Selective modification of proteins for pharmaceutical purposes is an increasingly important field. By selectively modifying proteins, for example by the addition of polyethyleneglycol (PEG), the pharmacological profile and half-life of proteins can be modified. In particular, it is desirable to generate long acting variants of proteins for use as pharmaceuticals. A number of techniques have been developed to selectively modify particular regions of proteins. Selective modification is particularly difficult for large proteins such as Factor VIII and Von Willebrand factor (vWF).
Proteins may be modified by reacting them with unselective reagents, which are capable of forming bonds to residues on the protein. A large range of different products will be formed by such methods, because the reagent can react with any suitable residue on the protein. An extension of this technique involves blocking specific sites on a protein with ligands that bind to these sites. The ligand/protein conjugate is then added to an unselective reagent that reacts with the unblocked sites on the protein, leaving the blocked sites unmodified.
Selective modification of proteins has also been investigated. One technique for selectively modifying proteins involves introducing cysteine residues as mutations at predefined sites in the protein. The mutated protein can then be reacted with a reagent that selectively reacts with the cysteine residues to give a selectively modified protein. Another technique involves adding a large substituent, typically PEG, directly to a protein by reacting the protein with a complex comprising the large substituent and a ligand that binds selectively to the protein.
Dawson et al, Journal of Thrombosis and Haemostasis, 5, 2062 to 2069 describes techniques for introducing labels into thrombin using targeting ligands and photocrosslinking reactions.
There is thus a need for more efficient and selective techniques for modifying proteins, particularly large proteins.